CN110353856B - Biological patch for posterior scleral reinforcement and preparation method thereof - Google Patents
Biological patch for posterior scleral reinforcement and preparation method thereof Download PDFInfo
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Abstract
A biological patch for reinforcing the back of eye and sclera is prepared from the heartbeats of different animals, such as cow, horse, pig, etc through removing impurities, degreasing, removing cells, carding, thinning, thickening, cross-linking, shaping, packing, sterilizing, and features high uniformity of thickness, controllable stretching and elongation, high biocompatibility and high degradability; the invention can greatly provide the usable area of the pericardial material, improve the qualified rate of the patch and reduce the production cost. The biological patch is purple blue and spherical fusiform, the spherical radius is more than 12.0mm, the constant-load stretching rate is less than or equal to 3%, the water content is more than 300%, the thickness is 0.10-0.65 mmm, the length is 28-55 mm, the width is 8-18 mm, and the biological patch is used for treating high myopia and retinal detachment and macular cleavage caused by the high myopia, and is reliable and effective for stopping development and restoring vision.
Description
Technical Field
The invention relates to an ophthalmic medical apparatus, in particular to a biological patch for posterior scleral reinforcement surgery for treating pathological high myopia and a manufacturing method thereof.
Background
The thickness of the posterior sclera of the eye is thinned and the intensity is weakened due to genetic and microenvironment infection factors, so that the eye axis is continuously prolonged and the negative refraction degree is continuously increased, so that the progressive aggravation of the high myopia is realized; continued expansion of the posterior sclera can also lead to retinal detachment, and as the course of the disease progresses, the patient is often blinded by ocular fundus lesions after adulthood. According to statistics, the incidence rates of myopia of the eyes of Chinese students at different stages of primary school, middle school and university are 30%, 50% and 70%, wherein 3-5% of people have pathological high myopia.
The current correction means for high myopia are as follows: the three methods can only correct vision and can not prevent the development of high myopia by wearing glasses, cutting cornea by laser and implanting artificial lens into anterior chamber belt lens; even the most advanced femtosecond lasers are not suitable for the treatment of high myopia exceeding 1200 ° (-12D), too thin cornea after laser ablation has the risk of blindness due to keratoconus formed by elevated ocular pressure and/or perforation occurring at the time of corneal disorder.
The etiology of pathologically high myopia is not clear, but rather genetic association, changes in scleral biomechanics due to infection of the intraocular microenvironment are also an important factor. During the whole course of pathological myopia, progressive increases in the ocular axis can be detected, and as a result of the expansion of the eye wall (sclera), the two regions, the region from the serrata to the equatorial region and the posterior scleral tissue, are first damaged (grape swelling), which in turn causes macular degeneration such as macular hemorrhage, fissure and peripheral retinal degeneration leading to retinal detachment leading to severe loss of vision and even blindness. Currently there is no effective treatment for pathological myopia, and most scholars consider posterior scleral reinforcement as the only effective treatment for pathological myopia. The posterior scleral reinforcement is to reinforce the posterior wall (posterior sclera) of the eyeball by an operation method to prevent the continued extension of the eye axis, so that the progressive damage of diopter and fundus is stopped, thereby achieving the purpose of preventing and treating myopia. Several decades of clinical practices prove that the posterior scleral reinforcement can effectively control the extension of the eye axis of pathological myopia, prevent the generation of choroidal neovascularization, repair the detached retina and macular cleavage, and improve the eyesight of patients. Clinical studies have shown that posterior scleral stiffening can effectively control the progression of pathological myopia. However, the effect of the surgery tends to decrease with time, which is one of the reasons why some ophthalmologists have disputed about the surgery.
It is currently believed that the therapeutic mechanism of posterior scleral stiffening is primarily mechanical reinforcement of the posterior sclera, and that the implant material will eventually merge with the recipient sclera to prevent the ocular axis from expanding and delay or block the progression of myopia. Promoting the formation of scleral neovascularization, forming a new vascular network of sclera, enhancing blood circulation, improving scleral and choroidal nutrition, and related action mechanisms and fusion conditions are mostly found in animal experiments. Surgical treatment is carried out on the high myopia of the patient due to retinal detachment, and after more than ten years of operation, the implanted allogenic scleral strip is observed to be mostly absorbed by necropsy, but residual allogenic sclera and fascia tissues can still be firmly adhered in partial areas of the original implanted allogenic sclera, and the separation is difficult; similar cases have been reported abroad, and scleral patches have been found to be still intact ten years after surgery; the long-term efficacy of posterior scleral reinforcement has long been controversial, and the choice of materials clearly plays an important role in posterior scleral reinforcement.
Common among the alternative reinforcement materials are: allogenic sclera, allogenic dura mater, autologous fascia lata, costal cartilage, decellularized allogenic dermis, allogenic umbilical cord, etc.; the non-biological materials mainly comprise: polyester fiber net (dacron net) and plasma breeding silica gel, gelatin sponge, polytetrafluoroethylene resin, etc.; the ideal reinforcing material should be; good biocompatibility, sufficient sources, easy cutting, easy preservation, long-term retention, no degradation by hosts, easy fusion with surface sclera, easy ingrowth of new blood vessels, thereby improving choroidal blood supply. From the above point of view, the non-biological material is not degraded, but it cannot fuse with the superficial sclera to make the new blood vessel not easy to grow in, so that the choroidal blood supply cannot be improved, thus limiting its application. The heterogeneous biological material, such as bovine pericardium, has wide sources, is beneficial to industrial production, has low cost and simple and convenient preservation, and has wide application prospect.
There are a great deal of clinical experiments and application researches, the bovine pericardium material is treated by the presently disclosed crosslinking technology, and histology proves that along with the extension of implantation time, the tissue structure is less clear, the gaps among collagen fibers are increased, the fiber arrangement is irregular, collagen becomes more loose, and the collagen has fracture and fusion; at month 36 of implantation, the collagen fibers had undergone a high amount of resorption. The result shows that the long-term curative effect of posterior scleral reinforcement is unsatisfactory in clinic, and the tensile elongation rate is increased due to the degradation of the patch.
Chinese patent CN 102525729B discloses a technical scheme for preparing a biological patch for human eye posterior scleral reinforcement by using pericardium or dura mater of cattle, horses and pigs, wherein the dura mater of animals is uneven, uneven in thickness is like walnut skin, blood vessels are distributed like veins, a proper surface area is difficult to find for operation, and the patch yield is extremely low;
the pericardial tissue structure of the animal consists of collagen fibers and elastic fibers, wherein the fibrils of the collagen fibers are arranged in parallel to form a coarser bundle, the triple helix of the collagen fibers consists of two alpha 1 chains and one alpha 2 chain, each alpha chain contains 1050 amino acid residues, and the triple helix consists of a repeated Gly-X-Y sequence. X-proline, Y-hydroxyproline or hydroxylysine residues; this sequence curls the alpha chain into the left-hand helix, containing 3 amino acid residues per turn. The three strands of such helices are then wound into right-handed supercoils with each other, i.e., procollagens. The collagen molecules are mutually and orderly arranged in a step-like manner through lateral covalent crosslinking to form fibrils with the diameters of 50-200 nm and the lengths of 150nm to several micrometers, and transverse lines with the intervals of 67nm are visible under an electron microscope.
The patch prepared by the technology and other disclosed technologies is continuous along with implantation time, collagen fibers on the surface layer of the patch are degraded by the lasting action of collagenase in host tissues, so that residues of collagen fibers on the inner layer of the patch are in a free naked state, host endothelial cells are induced to be immersed and covered, aseptic inflammatory reaction and immune reaction of the host tissues are caused, and the degradation speed of the patch is further accelerated; the host sclera cannot form effective fusion with the patch, a new reinforced sclera cannot be formed, the posterior sclera is continuously expanded after the patch is completely degraded, the postoperative long-term curative effect is reduced, the myopia is aggravated progressively, and the disease recurs.
The natural collagen fiber and elastic fiber are in a neat bending shape, can be extended and expanded by external force, and the disclosed technical scheme for preparing a biological patch for posterior scleral reinforcement by using a heterogeneous animal (pericardium) material is used for experiments, and the prepared biological patch material has the breaking elongation of about 30 percent, can not meet the biomechanical requirement of the biological patch for posterior scleral reinforcement, and also can not fully meet the requirement of long-term implantation of biomechanical and biological functional properties of the human eyeball wall (posterior sclera).
In contrast to the human eye sclera, the biomechanical requirements of the bioscope for posterior scleral reinforcement are: the elongation of the patch under allowable stress is less than or equal to 3 percent.
The pericardial material of animals (cattle, horses and pigs) is a hydrophilic biological material, has fiber orientation, controllable crosslinking degree, weak antigenicity, good biocompatibility, no rejection reaction after implantation, high affinity with cells, and can stimulate excellent characteristics of cell proliferation, differentiation, biodegradation and the like. After implantation into the human body, the peptides are degraded into amino acids, short peptides become source materials for constituting cells or are discharged outside the body through metabolic pathways (hydrophilic materials are important for attachment, growth and nutrient transfer of cells).
The presently disclosed technical solution has been to prepare bovine pericardium into heart valves for cardiac surgery; used for repairing meninges (trauma, brain surgery, repair to prevent subcutaneous adhesion to brain tissue, and prevent surgical epileptic occurrence); a sling for ophthalmic plastic- (ptosis); for general surgery-abdominal wall defects such as inguinal hernias.
The prior art cannot be used as a biological patch for posterior scleral reinforcement surgery due to failure in improving the elasticity and tensile elongation of the material and failure in eliminating the degradability of the material, and cannot guarantee the long-term curative effect for posterior scleral reinforcement surgery.
In view of the foregoing, there is a need for a new preparation method that overcomes the shortcomings of the prior art, and makes bovine pericardium a biological patch for posterior sclera, and provides a biological patch for posterior scleral reinforcement that is not weaker than the characteristics of human eye sclera, so as to meet the needs of many patients for treating high myopia.
Genipin is an iridoid heterocyclic compound, has a plurality of active functional groups such as hydroxyl, carboxyl and the like, is an excellent natural crosslinking agent, and can be crosslinked with protein, collagen, gelatin, chitosan and the like to prepare biological materials for clinical medical diagnosis and treatment processes, such as artificial bones, tissue repair, wound dressing materials and the like, and the toxicity of the genipin is far lower than glutaraldehyde (the toxicity of the genipin is one thousandth of glutaraldehyde) and other common chemical crosslinking agents. It also has antigen removing effect, and can react with free amino groups of residues such as lysine, hydroxylysine, arginine, etc. in collagen molecule. The cytotoxicity is low because the ring-shaped crosslinks formed by genipin are more stable than other network crosslinks and linear crosslinks. Genipin reacts with amino acid residues to form blue pigment, which may be produced by genipin and methylamine reactions, consisting of 40-44 monomers, suggesting that it can form a molecular crosslink of cyclic structure inside the collagen molecule; compared with other crosslinking methods, the genipin crosslinked collagen has the maximum thermal shrinkage Temperature (TS), elastic modulus and tensile strength; and after being implanted into a human body, calcification is not easy to occur. The toughness, mechanical strength, degradation resistance and other properties of the crosslinked biological film material are related to the crosslinking degree, and the toughness is increased, the mechanical strength is improved, the degradation resistance is improved, but the compliance is reduced as the hardness of the material is increased. The degree of crosslinking is related to the conditions of the crosslinking reaction, such as temperature, pH, concentration of the crosslinking agent, crosslinking time, etc.
Disclosure of Invention
The invention aims to provide a xenogeneic biological patch which is not weaker than the biomechanical and biological functional requirements of human eye sclera for posterior scleral reinforcement; and simultaneously provides a preparation method of the xenogenic biological patch for realizing the requirements.
The invention comprises the following steps: (1) preparing a blank by material selection: a. taking the pericardium of a mature cow, pig and horse which are qualified in fresh quarantine, removing impurities, fat and lacing, sterilizing and cleaning; b. removing fat by using a detergent, and flushing with physiological saline for 3 times; c. the damaged part which is obviously thickened at the inlet and outlet of the large blood vessel and is not suitable for application is sheared off; d. and (3) placing the prepared pericardium blank material into a disinfectant for storage for later use.
(2) Decellularized preparation of substrate: a. immersing the pericardium blank material into a decellularized solution for 2-8 h to destroy and dissolve the cell structure in the pericardium, b, washing the blank material subjected to the decellularization treatment with PBS solution for 3 times for 5min each time; c. repeating the steps a, b, three times to obtain the pericardial base material after cell removal.
(3) Arranging pericardium: a. taking the moderate part of the acellular pericardium base material, preparing a test standard sample, measuring and recording the breaking strength of the test standard sample; b. spreading the pericardial base material to be treated on a cross-linked bracket, and applying an outward acting force on the periphery of the blank material by taking the bracket as a support, wherein the acting force is 40-60% of pericardial fracture strength; the force value can lead the pericardium to be stretched to be in a completely flat state, and the pericardium has certain elasticity when touched by hands; c. obtaining a thickness topographic map of the material by using a digital thickness ruler or an electronic thickness scanner; d. taking the average thickness, and marking the edge of an ultra-thick area (> 30% average thickness) namely an ultra-thin area (< 30% average thickness); e. applying a local thermal denaturation treatment to the ultrathin zone: the material is thermally shrunken and denatured, and the thickness is increased to more than 1.5 times of the original thickness; f. carrying out local dehydration treatment on the ultra-thick region to enable the thickness of the region to be reduced by more than 20%, and simultaneously increasing the toughness, tensile property and pericardial material of the region to gradually show a semitransparent state from white; g. applying outward acting force around the material by taking the cross-linking bracket as a support, wherein the force value is 70% -95% of the breaking strength, stretching the material to enable the thickness of all areas to be consistent, and the error is less than +/-20%; h. the substrate was held under external tension on a cross-linked support. i. Attaching a shaping male die to the rough surface of the pericardium, attaching a shaping female die to the smooth surface of the viscera of the pericardium, and clamping the two dies up and down correspondingly and tightly, so as to clamp the pericardium completely; the shaping template is made of microporous material (such as sintered ceramic, microporous metal, foamed hard alloy or plastic) and has air-permeable and liquid-permeable effects, and can allow the crosslinking liquid to pass through the template and fully contact with pericardium to crosslink.
The shaping concave and convex mould plates are mutually corresponding and made of breathable materials, the surface of the convex mould plate is provided with at least one hemispherical bulge, and the spherical radius is between 12.0 and 18.0mm; the surface of the concave template is provided with at least more than one hemispherical pit, and the radius of the hemispherical pit is 0.2-0.60 mm larger than that of the spherical bulge of the convex template; and corresponds to the spherical convex surface of the convex mold plate.
(4) Preparing a refined material through crosslinking: a. completely immersing the cross-linking bracket and the substrate fixed by the concave-convex template into a decellularized solution, and cross-linking under shaking conditions; b. the crosslinking equipment parameters were: the temperature is 4-35 ℃; the time is 24-168 hours; the shaking angle is > +/-1 degrees; the swinging frequency is 1-100 times/min; c, crosslinking agent: the cross-linking agent is genipin solution with the concentration of 0.25-2.0%, and the solvent can be one of the following liquids: 1-75% alcohol, sterile deionized water or distilled water; d. the ratio of the pericardial base material to the cross-linking agent solution is 10-30% (W/V), and the PH value of the cross-linking agent solution is 5-14; e. observing the crosslinking process, wherein the pericardial substrate gradually reacts with the crosslinking agent to generate intermolecular crosslinking and intramolecular crosslinking between the collagen fiber and the elastic fiber, and the active functional group of genipin and the amino acid residue of the pericardial collagen fiber can generate crosslinking to form a polymer dark (purple blue) nontoxic pigment in the crosslinking process; the observation color is uniform and stable, the base material is increased, the strength is increased, and the toughness is increased; when the internal and external colors of the base material are consistent, the crosslinking is basically completed; f. after the crosslinking is finished, opening the upper and lower shaping concave-convex templates, taking off the pericardium from the crosslinking bracket, and generally observing: the base material should keep hemispherical, the surface is smooth and soft, and rebound, contracture and deformation phenomena after stretching and releasing are not occurred any more; so as to obtain the cross-linked pericardium finishing material, wherein the stretching elongation of the finishing material is less than or equal to 3 percent when the finishing material tolerates allowable stress of 20MPa and working stress of 12 MPa.
(5) Crosslinking post-treatment: a. repeatedly cleaning the cross-linked pericardium fine material which is complete and has no split and basically consistent thickness with distilled water for 3 times, each time for 3-5 min; b. and 5-30% glycine solution is used for carrying out neutralization reaction on the pericardium so as to eliminate residual cross-linking agent. c. Rinsing the pericardium extract material with distilled water for 3-5 times, each time for 3-5 min;
d. preparing a test sample according to the specification, and testing biomechanical properties of the cross-linked pericardium finishing material: the breaking strength is improved (28-68 MPa, different thickness is different) compared with that of the uncrosslinked material, and is not lower than 20MPa of allowable stress and is far higher than 12MPa of working stress; the stretching rate of the pericardium finishing material is less than or equal to 3 percent when the fixed load of the pericardium finishing material is 20 MPa; the water content is more than 300 percent.
(6) Packaging and sterilizing: a. and (3) forming: selecting a qualified spherical refined material with perfect thickness basically consistent and tested, inputting a three-axis three-dimensional laser cutting machine into a biological patch for posterior scleral reinforcement according to the requirement of clinical posterior scleral repair by using a spherical fusiform graph designed by a computer; b. and (3) packaging: placing the cut patch into a bubble cap container containing sterile normal saline for heat sealing and packaging, wherein the parameters and sealing performance of a packaging machine need to be verified and confirmed by a process, and the packaging mark and the code are required to meet the related regulation requirements; c. sterilizing, namely sterilizing the biological patch which is well packaged; the sterilization mode can be gamma rays, linear electron accelerators and ethylene oxide; the sterilization equipment, the process parameters and the final sterilization effect are verified and confirmed, so that the sterility level of the sterilization quality is ensured to meet the requirement of regulations; the sterilized biological patch for posterior scleral reinforcement is obtained.
The step (1) is to obtain materials, wherein the bovine pericardium sources are fresh bovine pericardium which is fed in a non-epidemic area with the age of 3-5 years, is qualified by quarantine and is slaughtered at fixed points; the source of the pig pericarp is to feed the fresh pig pericarp which is subjected to quarantine qualification and fixed-point slaughter in a non-epidemic area and is more than 1 year old.
The step (2) of the decellularized solution refers to: triton-100, detergent may be PBS solution; the decellularization method can also be performed with supercritical CO 2 The fluid vacuum device lyses off cells, or in a repeated freeze-thaw fashion.
In the step (3) of finishing: taking a bovine pericardium test sample to be treated by a universal electronic material tester according to a standard method, and measuring the breaking strength and the elongation of the bovine pericardium test sample; the cross-linking bracket is a ring-shaped or polygonal fastening bracket which can be clamped and can adjust tension, and the material for preparing the bracket is stable and does not react with a cross-linking agent; the tension can be adjusted by using a tension sensor or a tension meter, and can also be obtained by comparing the stretching ratios; the heat source for the thermal shrinkage denaturation treatment can be a direct heat source or a steam source, the treatment temperature is 60-100 ℃, the treatment time is 3 s-5 min, and the thermal deformation and shrinkage of the part of pericardial material are realized, and the thickness is increased to about 1.5 times of the original thickness of the part of pericardial material; the dewatering treatment method is one of the modes of using freezing, ventilating and moisture absorbing agent locally, and the dewatering rate is greater than 20%, and the thickness is reduced to 60-90% of the original thickness, and the error of the original thickness is less than +/-10%.
The crosslinking instrument comprises: the basic structure of the basic box body, the measurement and control unit with temperature setting and the heating element is provided with a stirring or shaking device, and the speed, the amplitude and the time of the stirring or shaking device can be set and adjusted; and a crosslinking pool.
The pH value of the cross-linking agent solution can be adjusted by using a buffer solution within the range of 5-14; the buffer may be one or more of the following: disodium hydrogen phosphate/sodium hydroxide; sodium chloride/sodium hydroxide; sodium dihydrogen carbonate/potassium dihydrogen phosphate; sodium carbonate/bicarbonate.
The pH value of the cross-linking agent solution is adjusted within the range of 5-14, and the color of the pericardium after being cross-linked can be correspondingly controlled to be changed from blue to purple.
Compared with the prior technical proposal, the invention has the following progress: (1) mapping the pericardium in a flat state to obtain an average thickness, and marking out an ultra-thick area, namely an ultra-thin area; respectively applying thermal shrinkage denaturation process treatment and dehydration process treatment to the ultra-thick area and the ultra-thin area; the overall thickness of the pericardial material tends to be consistent, the usable area of the material is greatly increased, and the qualification rate and the utilization rate of the material are improved; (2) testing the breaking strength of the pericardium after the pericardium is decellularized, calculating the maximum tensile strength of the pericardium when the pericardium is close to a molding state according to the breaking strength, and then applying acting force on the periphery of the pericardium according to the tensile strength value to realize the comb-straightening and stretching of the collagen fibers and the elastic fibers of the pericardium, so that the pericardium can be in an extending and straightening state; but also avoid the damage of too large tension to the pericardium (the damage of fiber entering the shaping area) caused by the discomfort of the tension; under the condition, the cross-linking treatment is carried out, a cross-linked structural network of procollagen under the condition that the collagen fibers are straight is reconstructed, and the natural rebound, contracture and deformation phenomena of the pericardium collagen fibers are eliminated, so that the cross-linked pericardium is smoother, smoother and has better comfortable texture, the key is that the tensile extensibility requirement of the posterior sclera can be met, the tensile extension rate of the fine material is less than or equal to 3% when the fine material tolerates allowable stress of 20MPa, and the tensile extension rate is less than or equal to 3% when the working stress of 12 MPa. (3) The pericardium is subjected to spherical shaping by using the concave-convex template, the spherical spindle-shaped patch is designed by using a computer according to the condition of a patient, and a triaxial (three-dimensional) laser cutting machine is used for shaping, so that the patch and the posterior sclera of the patient are tightly attached to each other, the expanded posterior sclera is favorably fastened and contracted, the fusion of the patch and the posterior sclera is accelerated, and the repair treatment of retina is promoted.
The invention treats the pericardial material by a thermal shrinkage denaturation/dehydration/carding/crosslinking method, can control the elasticity and the stretching rate of the crosslinked biological material (pericardium), and can also meet the biomechanical requirements of different medical application environments.
The pericardial material is treated by a thermal shrinkage denaturation/dehydration/carding/crosslinking method, the residues of the whole layer of procollagen of the base material can be furthest exposed, the whole layer crosslinking is realized by the crosslinking process treatment, the crosslinking degree is greatly improved by more than 90 percent, all the residues are thoroughly linked, and the extremely low degradation rate is obtained.
The invention can control the color of reactant (pericardium) by adjusting the PH value, is more striking according to the requirement, is easier to distinguish with peripheral tissues during operation and is convenient to operate.
Drawings
FIG. 1 is a diagram showing a structure of a normal optical microscope of tissue sections of fresh bovine pericardium in a natural state;
FIG. 2 is a diagram showing the structure of tissue sections of bovine pericardium after decellularization under a common optical microscope;
FIG. 3 is a diagram showing the structure of a tissue slice under a common optical microscope after the pericardium is subjected to decellularization, thermal shrinkage deformation thickening, drying thinning, collagen fiber carding and chemical crosslinking;
FIG. 4 is a schematic diagram of the appearance and morphology of a patch obtained by removing cells, thermally shrinking, deforming, thickening, drying, thinning, carding and chemically crosslinking collagen fibers and shaping and cutting pericardium in the invention;
fig. 5 is a schematic diagram showing the specific positional relationship of the biological patch of the present invention for posterior scleral reinforcement with a human eye sphere.
Detailed Description
The invention relates to a biological patch for posterior scleral reinforcement, which is purple or blue in appearance and is in a spherical fusiform shape, wherein the spherical bending radius of the biological patch is more than 12.0mm; the maximum length is 28-55 mm; 8-18 mm at the maximum width and 2.0-5.0 mm at the narrowest ends; the thickness is 0.10-0.65 mm; the outer surface of the soft spherical fusiform tablet is smooth and flat, and the inner surface is slightly rough and can be applied to the retrobulbar sclera; the breaking strength is more than 28MPa, the stretching rate of the fixed load (20 MPa) is less than or equal to 3%, and the water content is more than 300%.
The invention relates to a biological patch for an ophthalmic posterior scleral reinforcement surgery and a preparation method thereof, and an exemplary embodiment of the invention comprises the following steps of:
(1) preparing blank by taking materials
a. Drawing materials: fresh quarantine of pericardium of healthy mature cattle, pig, horse or sheep without diseases, removing impurities, fat and lacing, sterilizing and cleaning;
b. selecting materials: cutting off the part of the pericardium damaged part, which is obviously thickened and is not suitable for application, of the edge of the inlet and outlet of the large blood vessel;
c. degreasing: removing fat in pericardium by using a detergent, and then flushing with normal saline for 3 times; at the moment, the pericardium is touched to have obvious astringent feel and no slipping feel, so that a pericardium blank is formed;
d. and (3) disinfection: and (3) placing the prepared pericardium blank material into a disinfectant for disinfection for later use.
The detergent can be one or a mixture of several of the following applications: detergent, clear water and detergent diluent.
The disinfectant may be: one or more of chemical or biological agents: such as 0.5% peracetic acid, 100u/ml gentamicin saline solution, 100u/ml penicillin/streptomycin saline solution, 0.5% benzalkonium chloride, etc.
FIG. 1 is a tissue slice of pericardial blank material after the above steps: it can be seen that fibroblasts, mesothelial cells and adipocytes exist in a large amount of collagen fibers.
(2) Decellularized preparation of substrates
a. Immersing the pericardium blank into the decellularized solution for 2-8 h to completely destroy and dissolve the cell structure in the pericardium;
b. washing the blank subjected to the cell removal treatment with PBS solution for 3 times for 5min each time; eluting the cell residue fragments;
c. repeating the step a and the step b for three times to obtain the pericardial base material after cell removal.
As can be seen in FIG. 2, the large number of collagen fiber bundles in the pericardial tissue section are in a naturally curved state, the collagen fiber gap is increased, and the whole nucleus and organelle tissue are not seen.
(3) Carding base material fiber
a. Taking the moderate part of the acellular pericardium base material, preparing a test standard sample, measuring and recording the breaking strength of the test standard sample;
b. spreading the pericardial base material to be treated on a cross-linked bracket, and applying an outward acting force on the periphery of the blank material by taking the bracket as a support, wherein the acting force is 40-60% of pericardial fracture strength; the whole pericardium is stretched to be in a completely flat state, and the pericardium can be elastically expanded by hand contact;
c. obtaining a thickness topography of the pericardial substrate by using a continuous digital thickness ruler or an electronic thickness scanner;
d. taking the average thickness, marking the edges of the ultra-thick area (> 20% average thickness) and the ultra-thin area (< 20% average thickness);
e. firstly, applying local thermal denaturation treatment to an ultrathin area: the material is thermally shrunken and denatured, and the thickness is increased by more than 50 percent;
f. then carrying out local drying and dehydration treatment on the ultra-thick area to ensure that the thickness of the ultra-thick area is reduced by more than 20 percent, and meanwhile, the toughness and the stretching resistance are increased, and the pericardial material can gradually show semitransparent shape from white;
g. applying outward acting force around the material, wherein the force value is 80% -95% of the breaking strength, stretching the material to enable the thickness of all areas to be consistent, and the error is less than +/-10%;
h. holding the outward pulling force to fix the substrate on the cross-linked support;
i. then attaching the shaping convex template to the rough surface of the pericardium; attaching a shaping concave template to the smooth surface of the viscera of the pericardium, wherein the two templates correspond up and down and are clamped tightly with each other, so that the pericardium can be completely clamped in the shaping concave template; the shaping template is made of breathable microporous materials (such as sintered ceramics, microporous metals, foamed hard alloy or plastics, and the like) and can enable the crosslinking liquid to pass through the shaping template to fully contact with pericardium so as to generate crosslinking effect.
The carding method in the step (3) is as follows: taking a bovine pericardium test sample to be treated by a universal electronic material tester according to a standard method, and measuring the breaking strength and the elongation of the bovine pericardium test sample; the cross-linking bracket is a ring-shaped or polygonal fastening bracket which can be clamped and can adjust tension, and the material for preparing the bracket is stable and does not react with a cross-linking agent; the tension can be adjusted by using a tension sensor or a tension meter, and can also be obtained by comparing the stretching ratios; the heat source for carrying out thermal shrinkage denaturation can be a direct heat source or a steam heat source, the temperature is 60-100 ℃, the time is 3 s-5 min, the denaturation and shrinkage of the bovine pericardium material are carried out, and the thickness is increased to be about 1.5 times of the original thickness of the bovine pericardium material; the dehydration thinning treatment method can be to locally carry out freezing, ventilation and moisture absorption to ensure that the dehydration rate is more than 30 percent and the thickness is thinned to 70 to 90 percent of the original thickness at the position, and the error between the original thickness and the average thickness is controlled to be less than +/-10 percent.
The shaping concave-convex templates are mutually corresponding and made of breathable materials, at least one hemispherical bulge is arranged on the surface of the convex template, and the spherical radius is 12.0-18.0 mm; the surface of the concave template is provided with at least one hemispherical pit, and the radius of the hemispherical pit is 0.2-0.60 mm larger than that of the spherical surface of the convex template; and corresponds to the spherical convex surface of the convex mold plate.
(4) Cross-linking to prepare refined material
a. Completely immersing the cross-linking bracket and the substrate fixed by the concave-convex template into a decellularized solution, and cross-linking under shaking conditions;
b. the crosslinking (plant run) parameters are: crosslinking temperature: 4-35 ℃, and the crosslinking time is as follows: 24-168 hours; the shaking angle of the cross-linking bracket or the cross-linking agent is > +/-1 DEG; shaking frequency: 1-100 times/min;
c. crosslinking agent: the cross-linking agent is genipin solution with the concentration of 0.25-2.0%, and the solvent for dissolving genipin can be one of the following liquids: 1-75% alcohol solution, physiological saline, sterile deionized water or distilled water;
d. the ratio of crosslinking material (pericardial base material) to crosslinking agent solution is 10-30% (W/V); the pH value of the cross-linking agent solution: 5 to 14;
e. observing the crosslinking process: the pericardial base material and the crosslinking agent are subjected to chemical reaction, intermolecular crosslinking and intramolecular crosslinking are carried out between the collagen fiber and the elastic fiber, and in the crosslinking process, active functional groups of genipin and amino acid residues of the pericardial collagen fiber are crosslinked to form a polymer dark (purple blue) nontoxic pigment; observing the uniform and stable color of the base material, improving the luster and strength of the base material, and increasing the toughness; when the colors of the inner layer, the outer layer and the whole layer of the base material are consistent, the crosslinking is basically completed;
f. after the crosslinking is finished, opening the upper and lower shaping concave-convex templates, taking off the pericardium from the crosslinking bracket, and generally observing: the base material should keep hemispherical, the surface is smooth and soft, and rebound, contracture and deformation phenomena after stretching and releasing are not occurred. So as to obtain the cross-linked pericardium finishing material, wherein the stretching elongation of the finishing material is less than or equal to 3 percent when the finishing material tolerates allowable stress of 20MPa and working stress of 12 MPa.
The crosslinking instrument comprises: the basic structure of the basic box body, the temperature setting measurement and control unit and the heating element is arranged in the basic box body, and the basic structure is also provided with a stirring or shaking device, and the speed, the amplitude and the time of the stirring or shaking device can be set and adjusted; and a moderately sized cross-linked solution pool.
The PH value of the cross-linking agent can be adjusted within the range of 5-14, the pericardium essence material after cross-linking is blue when the PH is less than 8, and the pericardium essence material after cross-linking is (deep) purple when the PH is more than 8. The pH value can be adjusted by buffer solution; the buffer may be one or more of the following: disodium hydrogen phosphate/sodium hydroxide; sodium chloride/sodium hydroxide; sodium dihydrogen carbonate/potassium dihydrogen phosphate; sodium carbonate/bicarbonate.
(5) Crosslinking post-treatment:
a. rinsing: taking out the cross-linked pericardium extract material from the cross-linking machine, repeatedly rinsing with distilled water for 3 times, and 3-5 min each time;
b. and (3) neutralization: neutralizing the pericardium finishing material by using 5-30% glycine solution to eliminate residual cross-linking agent;
c. cleaning: rinsing pericardium extract material with distilled water for 3-5 times, each time for 3-5 min;
d. and (3) testing: taking a specified test sample, and testing biomechanical indexes of the cross-linked pericardium material: the breaking strength is obviously improved (28-68 MPa, different thickness are different) when the material is not crosslinked, and the material is not smaller than 20MPa of allowable stress and larger than 12MPa of working stress; the fixed load stretching rate is less than or equal to 3 percent; the water content is more than 300 percent.
FIG. 3 is an electron microscope scan of the structure of the fibers after decellularization of bovine pericardium, combing and crosslinking; the fibril bundles are straight and smooth, gaps among the fibril bundles are compact, and a certain amount of crosslinking and linking are carried out among the fibril bundles;
(6) sterilizing formed package
a. And (3) forming: selecting a qualified spherical refined material which is good and basically consistent in thickness and is tested, inputting a three-axis three-dimensional laser cutting machine into a biological patch for posterior scleral reinforcement by using a spherical spindle graph designed by a computer; see figure 4 for details; FIG. 4-1; (4) -2; (4) -3 is a schematic structural view, a schematic external surface view and a schematic internal surface view of the patch, respectively;
b. and (3) packaging: placing the biological patch into a bubble cap container containing sterile physiological saline for heat sealing and packaging, wherein parameters and sealing performance of a packaging machine need to be verified and confirmed by a process, and external marks and codes of the packaging material meet the requirements of related regulations;
c. and (3) sterilization: sterilizing the biological patch which is well packaged; the sterilization mode can be gamma rays, linear accelerators, chemical agents or ethylene oxide; the sterilization equipment, the process parameters and the final sterilization effect are verified and confirmed, so that the sterility level of the sterilization quality is ensured to meet the requirement of regulations; the sterilized biological patch for posterior scleral reinforcement is obtained.
In the step (1), the materials are obtained, and the sources of the hearts of the cattle and the horses are fresh hearts which are 3-5 years old and are subjected to quarantine inspection, qualification and fixed-point slaughter in a non-epidemic area; pig pericardium is a fresh pericardium which is fed in non-epidemic areas for more than one year, is qualified through quarantine inspection and is slaughtered at fixed points.
The decellularization method in the step (2) refers to: one of the following methods; tritonX-100, supercritical CO 2 fluid.
The crosslinking equipment is a basic structure comprising a temperature measurement and control circuit and a heating element, and is also provided with a timer, a stirring or shaking mechanism and a device with the speed and the amplitude capable of being set and adjusted.
The cross-linking agent is: pharmaceutical grade Genipin (Genipin), chemical formula: c (C) 11 H 14 O 5 White crystalline powder with a purity of 98%.
The pH value of the cross-linking agent solution ranges from 5 to 14, and the cross-linking agent solution can be adjusted through a buffer solution; the buffer may be one or more of the following: disodium hydrogen phosphate/sodium hydroxide; sodium chloride/sodium hydroxide; sodium dihydrogen carbonate/potassium dihydrogen phosphate; sodium carbonate/bicarbonate.
By changing the pH value of the cross-linking agent solution, the color of the pericardium after being cross-linked can be correspondingly controlled to be changed from blue to purple within the range of 5-14.
In clinical application, the positional relationship between the posterior scleral biological patch 1 and the retrobulbar polar optic nerve SN, the superior temporal vortex vein NS and the inferior temporal vortex vein NX of the present invention is shown in fig. 5; because the invention adopts the spherical fusiform patch structure, the patch can be fully and tightly attached to the retrobulbar sclera, and the loose and expanded retrobulbar sclera can be well fastened to retract uniformly, so that the retrobulbar sclera and the intrabulbar retina can be correspondingly and uniformly approximated and attached; compared with other flat biological patches, the biological patch with the shape has better attaching area and better fusion with posterior sclera and repair effect on retinal detachment.
Claims (3)
1. A biological patch for posterior scleral reinforcement, characterized in that: the biological patch is a purple blue and spherical fusiform sheet body with the length of 28-55 mm; 8-18 mm at the widest part; 3-5 mm at the narrowest part and 0.10-0.65 mm in thickness; the outer surface is smooth, and the inner surface is slightly rough; breaking strength is more than 28MPa; allowable stress is more than 20MPa, and the elongation rate is less than or equal to 3%; the working stress is more than 12MPa, and the elongation rate is less than or equal to 3%; the water content is more than 300 percent; sterile; the substrate of the biological patch is derived from the fresh pericardium of an animal;
the preparation method of the biological patch for posterior scleral reinforcement comprises the following steps:
1) Preparing blank by taking materials
a. Drawing materials: selecting fresh pericardium of healthy mature animals which are qualified by quarantine;
b. selecting materials: removing pericardial peripheral impurities, fat and lacing; cutting off the part of the damaged part, which is obviously thickened, of the edge of the inlet and outlet of the heart great vessel and is not suitable for application;
c. degreasing: removing fat in pericardium by using a detergent, and then flushing with normal saline for 3 times; at the moment, the touch pericardium has astringent feel and good slip-free feel, and becomes a pericardium blank;
d. and (3) disinfection: placing the prepared pericardium blank material into a disinfectant for standby;
2) Decellularized preparation of substrates
a. Immersing the pericardium blank into the decellularized solution for 2-8 h to completely destroy and dissolve the cell structure in the pericardium;
b. washing the decellularized blank with PBS solution for 3 times for 5min each time; eluting the cell residue fragments;
c. repeating the decellularizing step a and the washing step b for three times to obtain a pericardial substrate after decellularizing;
3) Carding base material fiber
a. Taking the moderate part of the acellular pericardium base material, preparing a test standard sample, measuring and recording the breaking strength of the test standard sample;
b. spreading the pericardial base material to be treated on a cross-linked bracket, and applying an outward acting force on the periphery of the pericardial base material by taking the bracket as a support, wherein the acting force is 40-60% of the pericardial fracture strength; the whole pericardium is stretched to be in a completely flat state, and the pericardium can be elastically expanded by hand contact;
c. obtaining a thickness topography of the pericardial substrate by using a continuous digital thickness ruler or an electronic thickness scanner;
d. taking the average thickness, and marking the edges of an ultra-thick area with the average thickness of more than 20% and an ultra-thin area with the average thickness of less than 20%;
e. firstly, applying local thermal denaturation treatment to an ultrathin area: the material is thermally shrunken and denatured, and the thickness is increased by more than 50 percent;
f. then carrying out local dehydration treatment on the ultra-thick area to ensure that the thickness of the ultra-thick area is reduced by more than 20 percent, and meanwhile, the toughness and the stretching resistance are increased, and the pericardial material can gradually show a semitransparent state from white;
g. applying outward acting force around the material, wherein the force value is 80% -95% of the breaking strength, stretching the material to enable the thickness of all areas to be consistent, and the error is less than +/-10%;
h. fixing the base material on the cross-linked bracket under the outward pulling force;
g. attaching the shaping convex template to the rough surface of the pericardium; attaching a shaping concave template to the smooth surface of the viscera of the pericardium, clamping and tightly closing the two templates correspondingly from top to bottom, and completely clamping the pericardium base material in the concave template;
the shaping template is made of breathable microporous materials, so that the crosslinking liquid can fully contact with pericardium through the shaping template to perform crosslinking;
in the step 3): taking a bovine pericardium test sample to be treated by using a universal electronic material tester according to a standard method, and measuring the breaking strength and the elongation of the bovine pericardium test sample;
the cross-linking bracket is a ring-shaped or polygonal fastening bracket which can be clamped and can adjust tension, and the material for preparing the bracket is stable and does not react with a cross-linking agent; the tension can be adjusted by using a tension sensor or a tension meter, and can also be obtained by comparing the stretching ratios; the heat source for carrying out thermal shrinkage denaturation can be a direct heat source or a steam heat source, the temperature is 60-100 ℃, the time is 3 s-5 min, so that bovine pericardium materials are denatured and shrunken, and the thickness is increased to be more than 1.5 times of the original thickness; the dehydration treatment method can be carried out by means of freezing, ventilation and moisture absorbing agents locally, so that the dehydration rate is more than 30%, the thickness is reduced to 70-90% of the original thickness, and the error between the original thickness and the average thickness is less than +/-10%;
the shaping concave-convex templates are mutually corresponding and made of breathable materials, at least one hemispherical bulge is arranged on the surface of the convex template, and the spherical radius is 12.0-18.0 mm; the surface of the concave template is provided with at least one hemispherical pit, and the radius of the hemispherical pit is 0.2-0.60 mm larger than that of the spherical surface of the convex template; and corresponds to the spherical convex surface of the convex template;
4) Cross-linking to prepare refined material
a. Immersing the crosslinked bracket and the fixed base material into the acellular hot solution completely, and crosslinking under shaking conditions;
b. the crosslinking parameters were: the temperature is 4-35 ℃ and the time is 24-168 hours; the shaking angle of the cross-linking bracket or the cross-linking agent is > +/-1 DEG; shaking frequency is 1-50 times/min, and pH value of the cross-linking agent solution is 5-14;
c. crosslinking agent: the cross-linking agent is genipin solution with the concentration of 0.25-2.0%, and the genipin solution can be one of the following liquids: 1-75% alcohol solution, physiological saline, sterile deionized water or distilled water; the pH value of the cross-linking agent can be adjusted within the range of 5-14.0 by using buffer solution;
d. the ratio of the pericardial base material to the cross-linking agent solution is 10-30% (W/V);
e. crosslinking results: the active functional group of genipin and the amino acid residue of the pericardial collagen fiber undergo a crosslinking reaction, so that intermolecular crosslinking and intramolecular crosslinking are generated between the pericardial base material collagen fiber and the elastic fiber;
after crosslinking, a polymer dark nontoxic pigment can be formed;
observing the uniform and stable color of the base material, and basically completing the crosslinking process when the colors of the inner layer, the outer layer and the whole layer of the base material are consistent;
f. after the crosslinking is finished, opening the upper and lower shaping concave and convex templates, taking off the pericardium from the crosslinking bracket, and generally observing: the base material should keep hemispherical, the surface is smooth and flexible, and rebound, contracture and deformation phenomena after stretching and releasing are not occurred any more;
so as to obtain the cross-linked pericardium finishing material, wherein the stretching elongation of the finishing material is less than or equal to 3% when the finishing material tolerates 20MPa of allowable stress and 12MPa of working stress;
the pH value can be adjusted by using disodium hydrogen phosphate/sodium hydroxide buffer solution to carry out the cross-linking agent solution; controlling the pH value of the cross-linking agent solution to be between 5 and 14, wherein the cross-linked pericardium finishing material is blue when the pH value is less than 8, and the cross-linked pericardium finishing material is purple when the pH value is more than 8;
5) Crosslinking post-treatment:
a. rinsing: taking out the cross-linked pericardium extract material from the cross-linking machine, repeatedly rinsing with distilled water for 3 times, and 3-5 min each time;
b. and (3) neutralization: neutralizing the pericardium finishing material by using 5-30% glycine solution to eliminate residual cross-linking agent;
c. cleaning: rinsing the pericardium refined material with distilled water for 3-5 times, each time for 3-5 min;
d. and (3) testing: taking a specified test sample, and testing biomechanical indexes of the cross-linked pericardium material: the breaking strength is obviously improved compared with that of the non-crosslinked material, is not lower than the allowable stress by 20MPa and is far higher than the working stress by 8-12 MPa; the fixed load stretching rate is less than 3% under working stress; the water content is more than 300 percent;
6) Sterilizing formed package
a. And (3) forming: selecting a qualified spherical refined material which is good and basically consistent in thickness and is tested, inputting a three-axis three-dimensional laser cutting machine into a biological patch for posterior scleral reinforcement by using a spherical spindle graph designed by a computer;
b. and (3) packaging: placing the biological patch into a bubble cap container containing sterile physiological saline for heat sealing and packaging, wherein parameters and sealing performance of a packaging machine need to be verified and confirmed by a process, and external marks and codes of the packaging material meet the requirements of related regulations;
c. and (3) sterilization: sterilizing the biological patch which is well packaged;
the sterilization mode can be gamma rays, linear accelerators, chemical agents or ethylene oxide; the sterilization equipment, the process parameters and the final sterilization effect are verified and confirmed, so that the sterility level of the sterilization quality is ensured to meet the requirement of regulations; the sterilized biological patch for posterior scleral reinforcement is obtained;
in the step 1), the beef and horse pericardium sources are fresh beef pericardium which is fed in a non-epidemic area with the age of 3-5 years and is subjected to quarantine inspection to be qualified and slaughtered at fixed points; the pig pericardium is a fresh pig pericardium which is fed in non-epidemic areas for more than one year, is qualified through quarantine inspection and is slaughtered at fixed points;
the step 2) cell removing solution refers to: one or more of the following solutions; tritonX-100, supercritical CO 2 A fluid;
the crosslinking equipment is a basic structure comprising a temperature setting, a temperature sensor, a measurement and control circuit and a heating element, and is also provided with a timer, a stirring or shaking device and a speed and amplitude of the device can be set and adjusted;
the cross-linking agent is: genipin (Genipin), chemical formula: c (C) 11 H 14 O 5 Purity 98%;
the pH value range of the cross-linking agent solution can be between 5 and 14, and the color of the cross-linked pericardium can be correspondingly controlled to be changed from blue to purple by adjusting the pH value of the cross-linking agent solution through buffer solution; the buffer may be one or more of the following: disodium hydrogen phosphate/sodium hydroxide; sodium chloride/sodium hydroxide; sodium dihydrogen carbonate/potassium dihydrogen phosphate; sodium carbonate/bicarbonate.
2. A biosome for posterior scleral reinforcement according to claim 1, wherein: the animals are healthy, mature and quarantined cattle, horses and pigs.
3. A biosome for posterior scleral reinforcement according to claim 1, wherein said patch is treated by selection, decellularization, thermal shrinkage, drying, carding, chemical crosslinking, with a degree of crosslinking of > 90%.
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| CN115463257B (en) * | 2022-09-13 | 2023-12-29 | 娜斯嘉生物科技(嘉兴)有限公司 | Composite reinforced scleral patch and preparation method thereof |
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